Cellulases consist of a complex enzyme system which catalyzes an enzymatic reaction, in which celluloses and analogous polysaccharides thereof are hydrolyzed to glucose, or cellobiose or cello-oligosaccharides, and are understood to be a general term for enzymes referred to as C.sub.1 enzymes, C.sub.x enzymes, .beta.-glucosidases, exo-.beta.-glucanases, endo-.beta.-glucanases, cellobiases or the like according to their action mechanisms. Viewed from a historic light, the research of the cellulases has been devotedly advanced for a long time with a view toward putting biomass resources to good use. Fungi, for example, the genera Trichoderma, Aspergillus, Acremonium and Humicola have been required for sources of their supply. However, cellulases from microorganisms including fungi have specificity on action of their constitutive enzyme groups and diversity on their physicochemical properties and the like. Therefore, their actual conditions can be yet hardly said to have been made clear.
Among the cellulases, those particularly high in action on carboxymethylcellulose, i.e., endo-type hydrolytic action, are called carboxymethylcellulases. In recent years, an application to, for example, an additive for laundry detergents has been developed as a new industrial use for the cellulases including carboxymethyl-cellulases. Judging from the above-described cellulases from microorganisms, however, most of cellulases produced from microorganisms in natural world show optimum activities at acidic pHs and are deactivated at alkaline pHs. Namely, they are acidic cellulases (optimum active pH: 4-6). Under the circumstances, there has been found only few alkaline cellulases and alkali-resistant cellulases, suitable for use in laundry detergent which gives alkaline pHs.
The term "alkaline cellulase" used herein is intended to mean one whose optimum pH is in an alkaline range, and the term "alkali-resistant cellulase" means one whose optimum pH is in a neutral to acidic range, but which has a satisfactory activity in an alkaline region as compared with an activity at an optimum pH and is maintained stable.
Namely, there have heretofore been reported only, as processes for the production of alkaline cellulases and alkali-resistant cellulases, which can be used in laundry detergent compositions, a process in which an alkaliphilic species of the genus Bacillus is incubated to collect Cellulase A (Japanese Patent Application Laid-Open No. 28515/1975), a process in which an alkaliphilic species belonging to the genus Cellulomonas is incubated to produce alkaline Cellulase 301-A (japanese Patent Application Laid-Open No. 224686/1983), a process in which alkaliphilic Bacillus No. 1139 is incubated to produce a carboxymethylcellulase [F. Fukumori, T. Kudo and K. Horikoshi, J. Gen. Microbiol., 131, 3339(1985)], and a process in which a species of the genus Streptomyces is used to produce an alkaline cellulase (Japanese Patent Application Laid-Open No. 19483/1986). None of these processes were suitable for use in industrial production by fermentation.
On the other hand, it has recently been reported that Bacillus sp. KSM-635 (FRI Deposition No. 8872), which is a species of alkaliphilic microorganisms, effectively produces a carboxymethylcellulase suitable for use in laundry detergent compositions and moreover, the carboxymethylcellulase can be provided with higher yield by suitably selecting incubation conditions, thereby permitting its commercial and industrial production by fermentation.
However, the carboxymethylcellulase from the above-described Bacillus sp. KSM-635 shows an optimum active temperature of about 40.degree. C. and is suitable for low-temperature washing. On the other hand, there is a demand for an enzyme capable of using in laundry detergents at higher temperatures. In addition, it is desired that a carboxymethylcellulase having high stability at higher pH values should be found because the detergency of detergent generally becomes enhanced at higher pH values.
Therefore, it is an object of this invention to provide a carboxymethylcellulase capable of acting even at high temperatures, broad in active pH range and in particular, good in stability at high temperatures and high pHs.
The present inventors have continued an extensive search with a view toward finding microorganisms capable of producing a carboxymethylcellulase from the natural world. As a result, it has been found that microorganisms collected from the soil in The Philippines and belonging to the genus Bacillus produce new Carboxymethylcellulase 5430 or 5812, which is useful as an additive for laundry detergent compositions, and Carboxymethylcellulases 5430 and 5812 are broader in active pH range than the conventional carboxymethylcellulases, excellent in thermal stability and in particular, good in stability at high pH values, leading to completion of the present invention.